Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

Inflammatory profiling of patients with familial amyloid polyneuropathy.

BACKGROUND: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control. METHODS: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients. RESULTS: We found that the levels of six markers were increased (TNF-α, IL-1ß, IL-8, IL-33, IFN-ß and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1ß and IL-10, suggesting that inflammation may take place before fibril deposition. CONCLUSIONS: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation.

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.

3'-nucleotidase/nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps.

Leishmaniasis is a widespread neglected tropical disease caused by parasites of the Leishmania genus. These parasites express the enzyme 3'-nucleotidase/nuclease (3'NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3'NT/NU in Leishmania survival of NET-mediated killing. Promastigotes of Leishmania infantum were cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups of Leishmania during interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3'NT/NU inhibitor. Inhibition of 3'NT/NU by 3'-AMP, 5'-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show that Leishmania infantum induces NET release and that promastigotes can escape NET-mediated killing by 3'-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.

Leishmania infantum Axenic Amastigotes Induce Human Neutrophil Extracellular Traps and Resist NET-Mediated Killing.

Neutrophils are multifaceted cells that, upon activation, release meshes of chromatin associated with different proteins, known as neutrophil extracellular traps (NETs). Leishmania amazonensis promastigotes and amastigotes induce NET release, and we have identified the signaling pathways involved in NET extrusion activated by promastigotes. Amastigotes maintain the infection in vertebrate hosts, and we have shown the association of NETs with amastigotes in human biopsies of cutaneous leishmaniasis. However, the interaction of amastigotes and neutrophils remains poorly understood. Our study aimed to characterize the pathways involved in the formation of NETs induced by axenic amastigotes from L. infantum, the causal agent of visceral leishmaniasis. Human neutrophils pretreated with signaling pathway inhibitors were incubated with amastigotes, and NET release was quantified in the culture supernatant. Amastigote viability was checked after incubation with NETs. We found that the release of NETs by neutrophils stimulated with these amastigotes requires the participation of elastase and peptidyl arginine deaminase and the involvement of PI3K, ROS, and calcium. Moreover, amastigotes are not susceptible to NET-mediated killing. Altogether, these findings improve our comprehension of the signaling pathways implicated in the interaction between amastigotes and human neutrophils.

Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.

Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania-neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.

A sand fly salivary protein acts as a neutrophil chemoattractant.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus.

Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

Immunity to vector saliva is compromised by short sand fly seasons in endemic regions with temperate climates.

Individuals exposed to sand fly bites develop humoral and cellular immune responses to sand fly salivary proteins. Moreover, cellular immunity to saliva or distinct salivary proteins protects against leishmaniasis in various animal models. In Tbilisi, Georgia, an endemic area for visceral leishmaniasis (VL), sand flies are abundant for a short period of ≤3 months. Here, we demonstrate that humans and dogs residing in Tbilisi have little immunological memory to saliva of P. kandelakii, the principal vector of VL. Only 30% of humans and 50% of dogs displayed a weak antibody response to saliva after the end of the sand fly season. Likewise, their peripheral blood mononuclear cells mounted a negligible cellular immune response after stimulation with saliva. RNA seq analysis of wild-caught P. kandelakii salivary glands established the presence of a typical salivary repertoire that included proteins commonly found in other sand fly species such as the yellow, SP15 and apyrase protein families. This indicates that the absence of immunity to P. kandelakii saliva in humans and dogs from Tbilisi is probably caused by insufficient exposure to sand fly bites. This absence of immunity to vector saliva will influence the dynamics of VL transmission in Tbilisi and other endemic areas with brief sand fly seasons.

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Gut Microbes Egested during Bites of Infected Sand Flies Augment Severity of Leishmaniasis via Inflammasome-Derived IL-1ß.

Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1ß (IL-1ß), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1ß in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1ß are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.

Neutrophil Extracellular Traps Reprogram IL-4/GM-CSF-Induced Monocyte Differentiation to Anti-inflammatory Macrophages.

Monocyte-derived dendritic cells (mo-DCs) are essential for the development of a Th1 protective immune response against Leishmania parasites. It is well known that IL-4 and GM-CSF drive differentiation of human monocytes to dendritic cells (DCs). Here, we investigate if neutrophil extracellular traps (NETs) disrupt this process. NETs-enriched supernatants, generated after human neutrophil activation by Leishmania promastigotes, were added to monocytes and differentiation monitored by expression of molecules associated with macrophage and DCs phenotypes, cytokine production, and parasite killing. We found that NETs addition to IL-4/GM-CSF-treated monocytes prevented then to fully differentiate into DCs. No effect was observed if NETs were treated with DNase or by filtering the traps. Moreover, NETs closely interact with monocytes and downregulate the expression of the IL-4 receptor, which in turn disrupts fully differentiation of monocytes into DCs. Neutrophil elastase inhibition rescues the monocytes to DCs differentiation. Monocytes cultured with IL-4/GM-CSF and NETs differentiated into macrophages, as observed by the increased expression of CD68, CD32, and CD163, and decreased expression of CD80. Moreover, NET addition to IL-4/GM-CSF-treated monocytes rendered these cells less efficient to kill Leishmania parasites. Altogether, our results show that NETs interfere with IL-4/GM-CSF driven differentiation, reprogramming the generation of mo-DCs to an anti-inflammatory macrophage.

The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B.

Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni2+ increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system.

Impact of insect salivary proteins in blood feeding, host immunity, disease, and in the development of biomarkers for vector exposure.

Functional genomic approaches based on expression of recombinant proteins linked to biochemical and disease model approaches resulted in the discovery of novel biological activities and the role some of these proteins play in disease transmission. Importantly, the expression of salivary proteins was recently shown to be affected by environmental factors and by the presence of the pathogen in the salivary gland. A practical application resulting from insect saliva research is the use of insect antigenic salivary protein as biomarkers of vector exposure in humans and animal reservoirs, an approach that is yielding interesting results in the field.

Impact of Insect Salivary Proteins in Blood Feeding, Host Immunity, Disease, and in the Development of Biomarkers for Vector Exposure.

Functional genomic approaches based on expression of recombinant proteins linked to biochemical and disease model approaches resulted in the discovery of novel biological activities and the role some of these proteins play in disease transmission. Importantly, the expression of salivary proteins was recently shown to be affected by environmental factors and by the presence of the pathogen in the salivary gland. A practical application resulting from insect saliva research is the use of insect antigenic salivary protein as biomarkers of vector exposure in humans and animal reservoirs, an approach that is yielding interesting results in the field.

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates.

Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly-exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4(+)IFN-γ(+) lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

Warifteine, an alkaloid purified from Cissampelos sympodialis, inhibits neutrophil migration in vitro and in vivo.

Cissampelos sympodialis Eichl is a plant from the Northeast and Southeast of Brazil. Its root infusion is popularly used for treatment of inflammatory and allergic diseases. We investigated whether warifteine, its main alkaloid, would have anti-inflammatory effect due to a blockage of neutrophil function. In vivo warifteine treatment inhibited casein-induced neutrophil migration to the peritoneal cavity but did not inhibit neutrophil mobilization from the bone marrow. Analysis of the direct effect of warifteine upon neutrophil adherence and migration in vitro demonstrated that the alkaloid decreased cell adhesion to P and E-selectin-transfected cells. In addition, fLMP-induced neutrophil migration in a transwell system was blocked by warifteine; this effect was mimicked by cAMP mimetic/inducing substances, and warifteine increased intracellular cAMP levels in neutrophils. The production of DNA extracellular traps (NETs) was also blocked by warifteine but there was no alteration on PMA-induced oxidative burst or LPS-stimulated TNF α secretion. Taken together, our data indicate that the alkaloid warifteine is a potent anti-inflammatory substance and that it has an effect on neutrophil migration through a decrease in both cell adhesion and migration.

ETosis: A Microbicidal Mechanism beyond Cell Death.

Netosis is a recently described type of neutrophil death occurring with the release to the extracellular milieu of a lattice composed of DNA associated with histones and granular and cytoplasmic proteins. These webs, initially named neutrophil extracellular traps (NETs), ensnare and kill microorganisms. Similarly, other cell types, such as eosinophils, mast cells, and macrophages, can also dye by this mechanism; thus, it was renamed as ETosis, meaning death with release of extracellular traps (ETs). Here, we review the mechanism of NETosis/etosis, emphasizing its role in diseases caused by protozoan parasites, fungi, and viruses.